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Loading Data

tsa_pH <- 
merge_TSA(raw_data_path = 
              c("/Users/billy/Desktop/SGS_Lab/TSA/TSAR_Files/CAFL_pH_20211203_20211203_162735_RawData_CAFL_pH_TSA_20211203.eds.txt",
                "/Users/billy/Desktop/SGS_Lab/TSA/TSAR_Files/CAFL_pH_3_20211206_162845_RawData_CAFL_pH_3.eds.txt"),
          analysis_file_path = 
              c("/Users/billy/Desktop/SGS_Lab/TSA/TSAR_Files/CAFL_pH_20211203_20211203_162735_AnalysisResults.txt",
                "/Users/billy/Desktop/SGS_Lab/TSA/TSAR_Files/CAFL_pH_3_20211206_162845_AnalysisResults.txt"))
head(tsa_pH)
##                        well_ID Well Protein      Ligand       Tm dTm D
## 1 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 2 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 3 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 4 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 5 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 6 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
##   Analysis Group Flags     Experiment File Name      condition_ID Well.Position
## 1           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 2           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 3           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 4           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 5           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 6           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
##   Temperature Fluorescence
## 1    86.14173     200369.2
## 2    86.26060     199525.2
## 3    86.32003     199102.9
## 4    86.37946     198680.3
## 5    86.20116     199947.3
## 6    86.49833     197835.2

To get the ID:

condition_IDs(tsa_pH)
## [1] "CA FL_Tris (pH 8)"    "CA FL_Tris (pH 6.8)"  "CA FL_Tris (pH 4)"   
## [4] "CA FL_NaP (pH 8)"     "Buffer_Tris (pH 8)"   "Buffer_Tris (pH 6.8)"
## [7] "Buffer_Tris (pH 4)"   "Buffer_NaP (pH 8)"
control_ID <- "CA FL_Tris (pH 8)"

Summary of Data

TSA_Tms(tsa_pH)
##           condition_ID   Avg_Tm       SD_Tm    Tm_ 1    Tm_ 2    Tm_ 3    Tm_ 4
## 1    CA FL_Tris (pH 8) 53.13616  0.09486345 53.13263 53.13263 52.98625 53.28607
## 2  CA FL_Tris (pH 6.8) 53.30693  0.11118899 53.13263 53.27901 53.42539 53.28607
## 3    CA FL_Tris (pH 4) 56.01484  0.24237114 55.76747 55.91385 55.76747 56.06704
## 4     CA FL_NaP (pH 8) 52.08727  7.39342995 54.45005 54.74281 54.88919 37.03931
## 5   Buffer_Tris (pH 8) 56.06023 45.74976773       NA 23.71026 88.41020       NA
## 6 Buffer_Tris (pH 6.8) 75.60417 26.06299696 92.21608 87.38554 24.29578 73.48473
## 7   Buffer_Tris (pH 4) 76.46719 29.50544327 24.00302 88.84934 92.65522 92.21973
## 8    Buffer_NaP (pH 8) 48.59927 30.71310609       NA       NA 29.41908       NA
##      Tm_ 5    Tm_ 6 Protein        Ligand
## 1 53.13970 53.13970   CA FL   Tris (pH 8)
## 2 53.43243 53.28607   CA FL Tris (pH 6.8)
## 3 56.35978 56.21341   CA FL   Tris (pH 4)
## 4 55.33521 56.06704   CA FL    NaP (pH 8)
## 5       NA       NA  Buffer   Tris (pH 8)
## 6 92.07336 84.16953  Buffer Tris (pH 6.8)
## 7       NA 84.60864  Buffer   Tris (pH 4)
## 8 84.02316 32.35556  Buffer    NaP (pH 8)

Box Plot of Data:

TSA_boxplot(tsa_pH,
            control = control_ID)
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TSA_boxplot(tsa_pH,
            control = control_ID,
            color_by = "Ligand")
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Normalize curves

normalized_tsa_pH <- normalize_fluorescence(tsa_data = tsa_pH)
head(normalized_tsa_pH)
##                        well_ID Well Protein      Ligand       Tm dTm D
## 1 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 2 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 3 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 4 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 5 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
## 6 A01_CAFL_pH_TSA_20211203.eds  A01   CA FL Tris (pH 8) 53.13263  <NA>
##   Analysis Group Flags     Experiment File Name      condition_ID Well.Position
## 1           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 2           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 3           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 4           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 5           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
## 6           AG 1     2 CAFL_pH_TSA_20211203.eds CA FL_Tris (pH 8)           A01
##   Temperature Fluorescence       RFU
## 1    86.14173     200369.2 0.1582415
## 2    86.26060     199525.2 0.1562010
## 3    86.32003     199102.9 0.1551799
## 4    86.37946     198680.3 0.1541584
## 5    86.20116     199947.3 0.1572215
## 6    86.49833     197835.2 0.1521150

Curves of each condition with all replicates shown

for(i in 1:condition_IDs(normalized_tsa_pH, n = TRUE)) {
    cond_i <- condition_IDs(normalized_tsa_pH)[i]
    subset_i <- normalized_tsa_pH$condition_ID == cond_i
    subset_i <- normalized_tsa_pH[subset_i, ]
    plot_i <- TSA_wells_plot(subset_i)
    
    print(plot_i)
}
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Plots to compare the control condition to:

Normalized Data

tsa_compare_plot(normalized_tsa_pH,
                 y = "RFU",
                 control_condition = control_ID)
## $`CA FL_Tris (pH 6.8)`

## 
## $`CA FL_Tris (pH 4)`

## 
## $`CA FL_NaP (pH 8)`

## 
## $`Buffer_Tris (pH 8)`

## 
## $`Buffer_Tris (pH 6.8)`

## 
## $`Buffer_Tris (pH 4)`

## 
## $`Buffer_NaP (pH 8)`

## 
## $`Control: CA FL_Tris (pH 8)`

Original Data

tsa_compare_plot(normalized_tsa_pH,
                 y = "Fluorescence",
                 control_condition = control_ID)
## $`CA FL_Tris (pH 6.8)`

## 
## $`CA FL_Tris (pH 4)`

## 
## $`CA FL_NaP (pH 8)`

## 
## $`Buffer_Tris (pH 8)`

## 
## $`Buffer_Tris (pH 6.8)`

## 
## $`Buffer_Tris (pH 4)`

## 
## $`Buffer_NaP (pH 8)`

## 
## $`Control: CA FL_Tris (pH 8)`

Functions to extract info from TSA data

# -- Condition IDs
condition_IDs(normalized_tsa_pH)
## [1] "CA FL_Tris (pH 8)"    "CA FL_Tris (pH 6.8)"  "CA FL_Tris (pH 4)"   
## [4] "CA FL_NaP (pH 8)"     "Buffer_Tris (pH 8)"   "Buffer_Tris (pH 6.8)"
## [7] "Buffer_Tris (pH 4)"   "Buffer_NaP (pH 8)"
# -- Well IDs
well_IDs(normalized_tsa_pH)
##  [1] "A01_CAFL_pH_TSA_20211203.eds" "A02_CAFL_pH_TSA_20211203.eds"
##  [3] "A03_CAFL_pH_TSA_20211203.eds" "A04_CAFL_pH_TSA_20211203.eds"
##  [5] "A05_CAFL_pH_TSA_20211203.eds" "A06_CAFL_pH_TSA_20211203.eds"
##  [7] "A07_CAFL_pH_TSA_20211203.eds" "A08_CAFL_pH_TSA_20211203.eds"
##  [9] "A09_CAFL_pH_TSA_20211203.eds" "A10_CAFL_pH_TSA_20211203.eds"
## [11] "A11_CAFL_pH_TSA_20211203.eds" "A12_CAFL_pH_TSA_20211203.eds"
## [13] "B01_CAFL_pH_TSA_20211203.eds" "B02_CAFL_pH_TSA_20211203.eds"
## [15] "B03_CAFL_pH_TSA_20211203.eds" "B04_CAFL_pH_TSA_20211203.eds"
## [17] "B05_CAFL_pH_TSA_20211203.eds" "B06_CAFL_pH_TSA_20211203.eds"
## [19] "B07_CAFL_pH_TSA_20211203.eds" "B08_CAFL_pH_TSA_20211203.eds"
## [21] "B09_CAFL_pH_TSA_20211203.eds" "B10_CAFL_pH_TSA_20211203.eds"
## [23] "B11_CAFL_pH_TSA_20211203.eds" "B12_CAFL_pH_TSA_20211203.eds"
## [25] "A01_CAFL_pH_3.eds"            "A02_CAFL_pH_3.eds"           
## [27] "A03_CAFL_pH_3.eds"            "A04_CAFL_pH_3.eds"           
## [29] "A05_CAFL_pH_3.eds"            "A06_CAFL_pH_3.eds"           
## [31] "A07_CAFL_pH_3.eds"            "A08_CAFL_pH_3.eds"           
## [33] "A09_CAFL_pH_3.eds"            "A10_CAFL_pH_3.eds"           
## [35] "A11_CAFL_pH_3.eds"            "A12_CAFL_pH_3.eds"           
## [37] "B01_CAFL_pH_3.eds"            "B02_CAFL_pH_3.eds"           
## [39] "B03_CAFL_pH_3.eds"            "B04_CAFL_pH_3.eds"           
## [41] "B05_CAFL_pH_3.eds"            "B06_CAFL_pH_3.eds"           
## [43] "B07_CAFL_pH_3.eds"            "B08_CAFL_pH_3.eds"           
## [45] "B09_CAFL_pH_3.eds"            "B10_CAFL_pH_3.eds"           
## [47] "B11_CAFL_pH_3.eds"            "B12_CAFL_pH_3.eds"
# -- Proteins Tested
TSA_proteins(normalized_tsa_pH)
## [1] "CA FL"  "Buffer"
# -- Ligands Tested
TSA_ligands(normalized_tsa_pH)
## [1] "Tris (pH 8)"   "Tris (pH 6.8)" "Tris (pH 4)"   "NaP (pH 8)"